Project Summary The composition of microorganisms that inhabit the gut, known as the gut microbiome, has been strongly associated with the pathogenesis of several diseases including cancer, diabetes, and multiple sclerosis. Notably, several high-profile studies have recently reported that the composition of the gut microbiome is correlated with responses to immune checkpoint inhibitors in human cancer patients. While these studies have proposed several mechanisms accounting for this phenomenon, a specific set of cellular and molecular interactions that explains these responses remains to be elucidated. One possible mechanism is through the immunologic recognition of the byproducts of microbial metabolism. Mucosal-associated invariant T cells (MAITs) are an abundant, highly-conserved subset of innate-like T cells that recognize products of microbial riboflavin synthesis presented by the non-polymorphic HLA-like molecule MR1. In animal models, mice grown in germfree conditions have a significant reduction in MAIT cells and the introduction of bacteria with high production of riboflavin drastically increases MAIT cell frequency and activation. Our preliminary data indicate that MAIT cells are found in human melanoma tumors. Furthermore, we found that patients with stage III/IV melanoma have significantly decreased frequencies of MAIT cells amongst their PBMCs, which can be reversed upon anti-PD1 immunotherapy treatment in responsive patients but not in non-responsive patients. Furthermore, we have found that the tumors of responding patients have increased expression of MR1 mRNA compared to non-responders. Interestingly, responsive patients also show increased relative abundance of riboflavin-synthesizing bacteria in their gut microbiome compared to non-responders. Two key questions arise from these observations. First, is the frequency or activation of MAIT cells driven by riboflavin-synthesizing bacteria in the gut microbiome? Second, do MAIT cells participate in anti-tumor immunity, and if yes, by what mechanism? Based on these observations, we hypothesize that 1) The frequency of activated MAIT cells is reflective of a microbiome enriched for riboflavin-producing species, and 2) MAIT cells possess anti-tumor activity, such that high MAIT cell frequency increases patient responsiveness to checkpoint immunotherapy. These hypotheses will be addressed by experiments in the following Specific Aims: (1) Determine if MAIT cell abundance and function are driven by riboflavin-synthesizing bacteria and whether this interaction influences clinical responses to anti-PD1 therapy; and (2) Determine the role of MAIT cells in anti-melanoma immunity. Should this exploratory study reveal a role for MAIT cells in anti-tumor immunity that is dependent on the contents of the microbiome, modalities aimed at expanding and/or activating MAIT cells during cancer therapy may provide substantial clinical benefit for cancer patients.